This invention is in the area of the prevention, diagnosis and treatment of autoimmune diseases, especially systemic lupus erythematosus.
Systemic lupus erythematosus (SLE) is similar to many other disorders in which autoantibodies are found and thought to be important in etiology and pathogenesis. SLE can be grouped with those diseases that commonly have autoantibodies present but for whom a central role of autoantibody in pathogenesis leading to clinical expression has yet to be fully established or accepted. Other such diseases include Sjogren""s syndrome, rheumatoid arthritis, juvenile onset diabetes mellitus, primary biliary cirrhosis, Wegener""s granulomatosis, inflammatory bowel disease, and many others.
Typically, autoimmune diseases present with a wide array of symptoms and clinical signs. The production of circulating autoantibodies to ribonucleoprotein complexes (RNPs) is a unifying characteristic of some of the rheumatic autoimmune diseases. The most common antigens in SLE and closely related disorders include: Ro/SSA, La/SSB, nRNP and Sm. Initially, these antibodies were found using double immunodiffusion, but more recently sensitive solid phase assays have been developed to quantitate the autoantibodies.
The Ro/SSA RNA-protein particle has been found to be a constituent of all human cells evaluated to date. Approximately half of Sjogren""s syndrome (SS) and systemic lupus erythematosus (SLE) patients have anti-Ro/SSA precipitins. Approximately 75% of patients with subacute cutaneous lupus erythematosus or complement component C2 deficiency with SLE have anti-Ro/SSA precipitins. Over 80% of mothers of newborns with neonatal lupus dermatitis or complete congenital heart block have this autoantibody. As many as 5% of patients with rheumatoid arthritis, polymyositis, and progressive systemic sclerosis have anti-Ro/SSA, as reported by R. M. Bernstein, et al., Mol. Biol. Med. 2:105-120 (1984); and J. B. Harley and K. K. Gaither, Autoantibodies. In Rheumatic Disease Clinics of North American: Systemic Lupus Erythematosus 14:1, 43-56. (1988).
Autoantibodies to the La/SSB ribonucleoprotein antigen are also found in patients with SS and SLE, as reported by Alspaugh, et al., Arthritis Rheum. 19:216 (1976) and Mattioli, et al., Arthritis Rheum. 17:421 (1974). In addition, these antibodies as reported by Horsfall, et al., J. Autoimmunity 4:165 (1991), thought to be pathogenic to the fetus during pregnancy in some mothers who have anti-La/SSB autoantibodies, where they are associated, along with anti-Ro/SSA, with complete congenital heart block (CCHB).
It has been an issue of intensive debate as to whether the many autoantibodies found in systemic lupus erythematosus and related diseases represent an antigen specific or a polyclonal, antigen non-specific response. Evidence that autoantibodies are important in the expression of SLE and related syndromes is convincing. Specific depletion in a heart block neonate (Harley, J. B., et al., Arthritis Rheum.28:1321-1325 (1985)) and specific anti-Ro/SSA immunoglobin deposition in human skin (Lee, L. A., et al., J. Clin. Invest. 83:1556-1562 (1989)) have been demonstrated. Specific concentration of anti-Ro/SSA has been shown in the immunoglobulin of renal eluates from kidneys affected by lupus nephritis (Maddison, P. J. and Reichlin, M. Arthritis Rheum. 22:858-863 (1979)). Anti-Ro/SSA has been found to be specifically concentrated in a parotid gland of a patient with Sjogren""s syndrome and primary biliary cirrhosis (Penner, E. and Reichlin, M. Arthritis Rheum. 25:1250-1253 (1982)). Observations that infants with transplacentally acquired maternal IgG develop neonatal lupus dermatitis and/or complete congenital heart block (Harley, J. B. and Gaither, K. K.: Autoantibodies. In Rheumatic Disease Clinics of North America: Systemic Lupus Erythematosus 14:1, 43-56 (1988)) strongly suggests that maternal autoantibody (anti-Ro/SSA or anti-La/SSB) transported across the placenta is a critical component required, but not sufficient, for these clinical problems.
The Ro/SSA family of proteins has now been shown to have several molecular forms which are operationally defined by the molecular weight of the antigen identified. A major form has an apparent molecular weight of 60 kiloDaltons (kD). This protein is associated with one of four hY RNAs. Recently, two additional proteins bound by anti-Ro/SSA sera have been identified by M. D. Rader, et al., J. Clin. Invest. 83:1556-1562 (1989), with molecular weights of 52 kD and 54 kD. A 48 kD protein, calmodulin, has been identified as being bound by anti-Ro/SSA sera (McCauliffe, et al., J. Clin. Invest. 85:1379-1391 (1990)). The La/SSB protein, a 48 kD peptide, as described by J. C. Chambers and J. D. Keene, Proc. Natl. Acad. Sci. USA 82:2115-2119 (1985), is also a member of this group of autoantibodies, and binds small RNAs with a polyuridine terminus, as reported by J. E. Stephano, Cell 36:145-154 (1984). La/SSB is bound by a third of the anti-Ro/SSA precipitin positive sera. The La/SSB protein has been purified from a variety of tissue sources and shown to be a 46 to 50 kD monomeric phosphoprotein, as reported by Habets, et al., EMBO J. 2:1625 (1983) and Venables, et al., Clin. Exp. Immunol. 54:731 (1983). It associates with RNA polymerase III transcripts, as reported by Lerner, et al., Proc. Natl. Acad. Sci. USA. 76:5495 (1979) and Steitz, et al., Cold Spring Harbor Symposium Quant. Biol. 47:893 (1983), and may function as a termination factor for this enzyme, as reported by Gottlieb, et al., EMBO J. 8:841 (1989). A nucleic acid dependent ATPase/dATPase enzymatic activity has also been attributed to La/SSB by Bachmann, et al., Cell 60:85 (1990).
Anti-Sm antibodies are frequently associated with SLE. These autoantibodies precipitate snRNP containing the U1, U2, U4/U6 and U5 RNA. These complexes form the spliceosome and splice heterogenous nuclear RNA, as reported by Sharp, Science 235:766 (1987) and Maniatis and Reed, Nature 325:673 (1987). Anti-Sm antibodies are directed against one or a combination of six polypeptides: B (26 kDa), Bxe2x80x2 (27 kDa), D (13 kDa), E/F (11 kDa doublet) and G (less than 10 kDa).
Nearly all rheumatic disease patients who form an anti-Sm precipitin in ouchterlony immunodiffusion have or eventually develop an anti-nRNP precipitin, as reported by Fisher, et al., Arthritis Rheum. 28:1348 (1985). Anti-Sm and anti-nRNP precipitins form a line of partial identify in Ouchterlony immunodiffusion, as discussed by Mattioli and Reichlin, J. Immunol. 110:1318 (1973). The basis for this partially shared antigenicity is explained by the composition of the U snRNP particles. The antigen for the anti-nRNP precipitin are the 70 kD, A, and C peptides that are unique to the U1 snRNP, B/Bxe2x80x2 and D peptides are also found on the U1 snRNP. The B/Bxe2x80x2 and D Ag, but not the 70 kDa, A or C, are found in the U2, U4/U6 and U5 snRNP. Hence, both anti-Sm and anti-nRNP bind anti-U1 snRNP activity, but only anti-Sm binds U2, U4/U6, and U5 snRNP.
U.S. Ser. No. 07/648,205 filed Jan. 31, 1991 by John B. Harley for xe2x80x9cAssays and Treatments for Autoimmune Diseasesxe2x80x9d, and U.S. Ser. No. 07/472,947 entitled xe2x80x9cAssays and Treatments for Autoimmune Diseasesxe2x80x9d filed Jan. 31, 1990, described a specific method to identify the etiologic or antigenic agent responsible for the production of autoantibodies characteristic of a particular disorder. The antigen is first isolated, using, for example, autoantibodies isolated from one or more patients. The antigen is then divided into overlapping short amino acid sequences, preferably twenty amino acids or less, most conveniently octapeptides. The sequences having the greatest reactivity with the autoantibodies are identified and then compared with all known amino acids sequences using the available computer data bases. The protein having the maximum number or proportion of sequences homologous to the sequences of greatest reactivity with the autoantibodies is among the likeliest candidate of the known sequenced proteins for the etiological agent or immunogen. Once the etiological agent and antigenic sequences are known, it is possible to design assays and reagents for the diagnosis and treatment of patients having either the etiological agent and/or autoantibodies.
The examples in the earlier applications used peptides derived from the sequence for the 60 kDa Ro/SSA protein and La/SSB, which were reactive with antisera from SLE and SS patients.
It is therefore an object of the present invention to provide additional diagnostic reagents for identifying and classifying individuals previously exposed to a particular immunogen or expressing autoantibodies reactive with Ro/SSA, La/SSB, nRNP, or Sm B/Bxe2x80x2 polypeptides, or the epitopes (or their immune equivalent) eliciting production of the autoantibodies.
It is a still further object of the present invention to provide methods and compositions for identifying and treating autoimmune disorders, such as Systemic Lupus Erythematosus and Sjogren""s syndrome.
A number of octapeptides have been generated from the sequences encoding the 60 kDa Ro/SSA peptide, the La/SSB autoantigen, the 70 kD nuclear ribonucleoprotein (nRNP), and the Sm B/Bxe2x80x2 polypeptide, which represent linear epitopes for autoantibodies present in the sera of SLE and SS patients.
For example, the most important antigenic peptides derived from Sm B/Bxe2x80x2 are (29) GTFKAFDK (SEQ ID NO:1), (45) CDEFRKIKPKNAKQP (SEQ ID NO:2), (94) RVPLAGAA (SEQ ID NO:3), (101) AGGPGVGRAAGRGVPAG (SEQ ID NO:4), (125) AGLAGPVRGVGGPSQ (SEQ ID NO:5), (140) QVMTPQGRGTVA (SEQ ID NO:6), (165) PTQYPPGRGTPPPPV (SEQ ID NO:7), (174) TPPPPVGRATPPPGI (SEQ ID NO:8), (184) PPPGIMAP (SEQ ID NO:9), (189) MAPPPGMRPPM (SEQ ID NO:10), (202) PIGLPPARGTPIGMPP (SEQ ID NO:11), (212) PIGMPPP (SEQ ID NO:12), (221) RPPPPGIRGPP (SEQ ID NO:13), and (228) RGPPPPGMRPPR (SEQ ID NO:14). Additional reactive peptides can be derived from (30) TFKAFDKHM (SEQ ID NO:15), (83) EGPPPKDT (SEQ ID NO:16), (88) KDTGIARV (SEQ ID NO:17), AND (120) IPQAPAGLAG (SEQ ID NO:18). These were determined by binding studies. PPPGMRPP (SEQ ID NO:123) is especially antigenic and is repeated three times in the sequence of B/Bxe2x80x2. The antigenicity of other peptides was determined and include the shorter peptides PPPGMRP (SEQ ID NO:124) and PPPGMR (SEQ ID NO:125). Substitution studies were also done. All 19 of the other common naturally occurring amino acids are substituted for an amino acid in a particular position. For example, the arginine in position six of PPPGMRPP (SEQ ID NO:123) can be substituted with: F, G, H, I, K, S, T, V, W and Y.
These peptides are useful in solid phase assays for patients characterized by the presence of these autoantibodies, and can be used to categorize patients as to the likelihood of developing certain conditions associated with SLE. The peptides are also potentially useful in treatment of these patients using immobilized peptide to remove aujtoantibody, to block binding of the autoantibodies with patient molecules reactive with the autoantibodies, or as a component of a vaccine.